Synthetic penicillins



United States Patent C) 3,352,852 SYNTHETKC PENICHLLENS Lee (I. Cheney,Fayetteville, N.Y., assignor, by mesne assignments, to Beecham GroupLimited, Brentford, Middlesex, England, a corporation of Great Britainand Northern Ireland No Drawing. Application Dec. 20, 1962, Ser. No.245,982, which is a division of application Ser. No. 847,874, Get. 22,1959. Divided and this application Feb. 14, 1966, Ser. No. 527,111

3 Claims. (Cl. 260-2391) This application is a continuation-in-part ofmy prior, copending application Ser. No. 815,287 filed May 25, 1959, andis also a division of my prior, copending application Ser. No. 245,982filed Dec. 20, 1962, now abandoned which was in turn a division of myprior, then copending application Ser. No. 847,874 filed Oct. 22, 1959,now abandoned as a continuation-in-part of said application Ser. No.815,287 and now abandoned.

This invention relates to new synthetic compounds of value asantibacterial agents, as nutritional supplements in animal feeds, asagents for the treatment of mastitis in cattle and as therapeutic agentsin poultry and animals, including man, in the treatment especially ofinfectious diseases caused by Gram-positive bacteria and, moreparticularly, relates to substituted6-(2-phenoxypropionamido)penicillanic acids and nontoxic salts thereof.

Antibacterial agents such as benzylpenicillin have proved highlyelfective in the past in the therapy of infections due to Gram-positivebacteria but such agents suffer from the serious drawbacks of beingunstable in aqueous acid, e.g. upon oral administration, and of beingineffective against numerous so-called resistant strains of bacteria,e.g. penicillin-resistant strains of Staphylococcus aureus(illicrococcus pyogenes var. aureus). In addition, benzylpenicillin isnot an effective agent against many bacteria which producepenicillinase. Many of the compounds of the present invention, inaddition to their potent antibacterial activity, exhibit resistance todestruction by acid or by penicillinase or are effective againstbenzylpenicillin-resistant strains of bacteria or inhibitbenzylpenicillinase and thus potentiate the action of benzylpenicillinwhen admixed therewith.

There is provided, according to the present invention, a member selectedfrom the group consisting of an acid having the formula wherein R R andR are each members selected from the group consisting of hydrogen,nitro, amino, (lower) alkylamino, di(lower)alkylamino, acylamino (wherethe acylating acid is an aliphatic carboXylic acid containing from oneto ten carbon atoms inclusive and the substituent may thus also be named(lower) alkanoylamino), (lower) alkyl, chloro, bromo, iodo,(lower)alkoxy, hydroxy, sulfamyl, benzyl and trifluoromethyl and Rrepresents a member selected from the group consisting of lower alkyl(including, both here and above, straight and branched chain saturatedaliphatic groups containing from one to ten carbon atoms inclusive),phenyl and phenylalkyl (including ,benzyl and aand B-phenethyl and oc-,B- and -phenylpropy1) and nontoxic salts thereof, including nontoxicmetallic salts such as sodium, potassium, calcium and aluminum, theammonium salt and substituted ammonium salts, e.g., salts of suchnontoxic amines as trialkylamines, including triethylamine, procaine,dibenzylamine, N-benzyl-beta-phenethylamine, l-ephenamine, N,

3,352,852 Patented Nov. 14, 1957 "ice N-dibenzylethylenediamine,dehydroabietylamine, N,N'- bis-dehydroabietylethylenediamine, and otheramines which have been used to form salts with benzylpenicillin. Alsoincluded within the scope of the present invention are easily hydrolyzedesters which are converted to the free acid form by chemical orenzymatic hydrolysis.

The products of the present invention are prepared by reaction of6-aminopenicillanic acid, preferably in the form of a neutral salt suchas the sodium salt or the triethylamine salt, with an acid chloridehaving the formula wherein R R R and R have the meaning set forth above,or its functional equivalent as an acylating agent for a primary aminogroup. Such equivalents include the corresponding carboxylic acidbromides, acid anhydrides and mixed anhydrides with other carboxylicacids, including monoesters, and particularly lower aliphatic esters, ofcarbonic acid.

Thus, an elegant procedure for preparing a compound of the presentinvention by way of a mixed anhydride with ethoxyor iscbutoxy-carbonicacid comprises mixing 0.01 mole of an acid (whose acid chloride is setforth above), 0.01 mole isobutyl chloroformate and 0.011 mole tertiaryhydrocarbonyl or aliphatic amine such as triethylamine in an anhydrous,inert and preferably water-miscible solvent such as p-dioxane (e.g., 20ml.) and if desired 2 ml. pure, dry acetone for about thirty minutes inthe cold, e.g., at about 4 C. To this solution of the mixed anhyd-ridethere is then added a chilled solution of 0.01 mole 6- aminopenicillanicacid and 0.01 mole tertiary hydrocarbonyl amine, e.g., triethylamine,in, for example, 20 ml. of a solvent such as water. The reaction mixtureis stirred for a period of an hour or so to form the substitutedammonium salt of the desired product. The mixture may then, if desired,be extracted at alkaline pH (such as pH 8; aqueous sodium bicarbonatemay be used, for example, if necessary to adjust the pH) with awater-immiscible solvent such as ether to remove unreacted startingmaterials. The product in the aqueous phase is then converted to thefree acid, preferably in the cold under a layer of ether by the additionof dilute mineral acid, e.g., 5 N H 50, to pH 2. The free acid is thenextracted into a water-immiscible, neutral organic solvent such as etherand the extract is washed with water quickly in the cold, if desired,and then dried, as with anhydrous N21 SO The product in the etherealextract in its free acid form is then converted to any desired metal oramine salt by treatment with the appropriate base, e.g., a free aminesuch as procaine base or a solution of potassium 2-ethylhexanoate in dryn-butanol. These salts are usually insoluble in solvents such as etherand can be recovered in pure form by simple filtration.

Another method of preparing an ethereal solution of the acid form of acompound of the present invention comprises preparing a solution in 20ml. of water of 0.00463 mole 6-aminopenicillanic acid and 1.56 gm.sodiurn bicarbonate, adding 0.00476 mole of an acid chloride whoseformula is set forth above and shaking vigorously at room temperature,e.g., for twenty to sixty minutes. The mixture is then extracted withether to remove unreacted or hydrolyzed starting materials. The solutionis then acidified (preferably in the cold) to pH 2, as with dilutesulfuric acid, and the free acid form of the product is extracted intoether (e.g., two portions of 25 ml.). This ethereal extract is dried, aswith anhydrous sodium sulfate, and the drying agent is removed to leavea dry ethereal solution from which the product is easily isolated,preferably in the form of an ether-insoluble salt such as the potassiumsalt. This procedure is used when the acid chloride reacts with aprimary amine more rapidly than it does with water, as determined bysimple test. In this procedure the acid chloride may be replaced by anequimolar amount of the corresponding acid bromide or acid anhydride.

Since some of the antibiotic substances obtained, by the process of thisinvention are relatively unstable compounds which readily undergochemical changes resulting in the loss of an antibiotic activity, it isdesirable to choose reaction conditions which are sufficiently moderateto avoid their decomposition. The reaction conditions chosen will, ofcourse, depend largely upon the reactivity of the chemical reagent beingused. In most instances, a compromise has to be made between the use ofvery mild conditions for a lengthy period and the use of more vigorousconditions for a shorter time with the possibility of decomposing someof the antibiotic substance.

The temperature chosen for the process of preparation of the derivativesof penicillanic acid should in general not exceed 30 C. and in manycases a suitable temperature is ambient temperature. Since the use ofstrongly acid or alkaline conditions in the process of this inventionshould be avoided, it has been found preferable toperform the process ata pH of from 6 to 9, and this can conveniently be achieved by using abuffer, for example a solution of sodium bicarbonate, or a sodiumphosphate buffer. In addition to the use of aqueous media for thereaction, including filtered fermentation broths or aqueous solutions ofcrude 6-aminopenicillanic acid, use can be made of organic solventswhich do not contain reactive hydrogen atoms. Examples of such inertsolvents are dimethylformamide, dimethylacetamide, chloroform, acetone,methyl isobutyl ketone and dioxane. Frequently it is highly satisfactoryto add an aqueous solution of a salt of 6-aminopenicillanic acid to asolution of the acylating agent in an inert solvent and preferably in aninert solvent which is miscible with Water, such as acetone ordimethylformamide. Vigorous stirring is of course advisable when morethan one phase is present, e.g., solid and liquid or two liquid phases.

At the conclusion of the reaction, the products are isolated if desiredby the techniques used with benzylpenicillin andphenoxymethylpenicillin. Thus, the product can be extracted into diethylether or n-butanol at acid pH and then recovered by lyophilization or byconversion to a solvent-insoluble salt, as by neutralization with annbutanol solution of potassium Z-ethylhexanoate, or the product can beprecipitated from aqueous solution as a water-insoluble salt of an amineor recovered directly by lyophilization, preferably in the form of asodium or potassium salt. When formed as the triethylamine salt, theproduct is converted to the free acid form and thence to other salts inthe manner used with benzylpenicillin and other penicillins. Thus,treatment of such a triethylamine compound in water with sodiumhydroxide converts it to the sodium salt and the triethylamine may beremoved by extraction, as with toluene. Treatment of the sodium saltwith strong aqueous acid converts the compound to the acid form, whichcan be converted to other amine salts,

e.g., procaine,,by reaction with the amine base. Salts so formed areisolated by lyophilization or, if the product is insoluble, byfiltration. A particularly elegant method of isolating the product as acrystalline potassium salt comprises extracting the product from anacidic, aqueous solution (e.g., pH 2) into diethyl ether, drying theether and adding at least one equivalent of a solution of potassiumZ-ethylhexanoate (e.g., 0.373 gm./ml.) in. dry n-butanol. The potassiumsalt forms, precipitates, usually in crystalline form, and is collectedby filtration or decantation.

When an acid chloride, an acid bromide or an acid anhydride is used in aprocess of the present invention, it is prepared from the correspondingacid according to the techniques set forth in the literature for acidssuch as phenylacetic acid and phenoxyacetic acid. In any instances wherethe substituted a-phenoxyalkanoic acid has not been described, it isprepared from the appropriately substituted phenol and the appropriatea-chloroor a-bromo-acid by the methods described in the art forphenoxyacetic acid or substituted phenoxyacetic acids or described orreferred to in US. Patents 2,854,450 and 2,479,297.

Preparation of u-aryloxyalkanoic acids In the preparation ofu-aryloxyalkanoic acids, four different methods have been used: A, B, Cand D.

Methods A and B are believed to be general for the preparation ofa-aryloxyalkanoic acids from unhindered phenols and u-haloalkanoicacids. The a-bromoalkanoic acids are more frequently used, but thea-chloroalkanoic acids are found to work equally Well. Of the twomethods, A is to be preferred over B.

When a-halo esters are used instead of a-haloalkanoic acids,and wherethe phenols are highly hindered, Methods C and D should be used. Oftenthe reaction time is shorter and the yields higher when C and D are usedfor the preparation of unhindered a-aryloxyalkanoic acids from thecorresponding a-halo esters. It is interesting to note that in cases ofvery highly hindered phenols, as in 2,6-diisopropylphenol, Method Cgives the best result.

No attempt was made to find conditions for optimum yields except in the.preparation of a-phenoxypropionic acid, where a better yield is obtainedwhen excess phenol is used (Method A).

In the recrystallization of the acids most of the common organicsolvents have been tried. It was found that isopropyl alcohol-Water,benzene-lower alkanes (Skelly C or B) work best for most of the acids.One recrystallization from these mixed solvents is usually enough togive a product of analytical purity, provided the starting materials areof good quality. In cases where the acids cannot be crystallized, theyare distilled under high vacuum.

The details of these four methods are as follows:

Method A.0.4 mole of phenol (it was found that a better yield wasobtained in the preparation of a-phenoxypropionic acid when an excess ofphenol was used) and 0.2 mole of an a-haloalkanoic acid weredissolvedtogether in ml. ethanol and an ice-cooled solution of 0.5 molesodium hydroxide in 30 ml. water was carefully added. The mixture wasthen heated under reflux on a steam bath for 22 hours.

The ethanol was completely removed under reduced pressure. The residuewas transferred to an Erlenmeyer flask containing 200 ml. of water, anddilute hydrochloric acid was added until the solution was strongly acid(pH 2). The precipitated acid was then taken up in ether and extractedwith a 5% sodium bicarbonate solution, which was then heated gently on asteam bath under reduced pressure to remove the dissolved ether. Onacidification with dilute hydrochloric acid in excess, the phenoxy-acidwas obtained as a crystalline precipitate. The yield of the crude acidwas usually 50-85% of the theoretical value. One recrystallization froman appropriate solvent usually gave a product of analytical purity.

Method B.To 0.5 mole of an u-haloalkanoic acid was added slowly in anice bath 0.5 mole sodium hydroxide in ml. H O.

A phenol (0.5 mole) was dissolved in a cooled solution of 0.5 molesodium hydroxide in 125 ml. H O. To this'sodium phenolate solution wasadded all at once the aqueous solution of the acid salt and the mixturewas heated on a steam bath for 20 hours. The solution was cooled to roomtemperature, strongly acidified wtih dilute hydrochloric acid, cooled inan ice bath for an hour and the precipitated oily acid was dissolved inether. The ether layer was separated and extracted with a 5% sodiumbicarbonate solution, acidified with an excess of dilute hydrochloricacid, and the crystalline precipitate was collected by suction. Onerecrystallization from an appropriate solvent would usually give aproduct of analytical purity. V

Method C.Into a 1-liter 3-necked flask equipped with a mechanicalstirrer, a reflux condenser above a Dean- Stark water trap, and adropping funnel were placed 0.2 mole of a phenol, 250 ml. of toluene and0.25 mole of flake sodium hydroxide (Mallinckrodt). The stirred mixturewas heated to boiling and maintained under reflux until 3.3 ml. of water(theory 3.6 ml.) had collected in the trap. Ethyl a-haloalkanoate (0.025mole) was then added dropwise at such a rate that the reaction wasmaintained under moderate reflux. The mixture was then boiled overnightunder reflux.

The mixture was cooled below C. and ml. of methanol and a solution of0.3 mole of sodium hydroxide in ml. of water were added consecutively.To complete the saponification the stirred mixture was heated undervigorous reflux for two hours. The hot alkaline mixture was then dilutedby the addition of 250 ml. of water. The alkaline aqueous layer wasseparated and strongly acidified with dilute sulfuric acid. Theprecipitated oily acid was caused to crystallize by chilling in an icebath while stirring the mixture with a glass rod. The product wascollected by suction and washed with 50 ml. of cold Water. Onerecrystallization from an appropriate solvent was usually enough toafford a product of analytical purity, provided the starting materialswere of good quality.

Method D.A solution of 0.156 mole of sodium, 0.156 mole of ethyla-haloalkanoate and 0.156 mole of a phenol in 120 ml. absolute ethanolwas refluxed for four hours. Then 120 ml. of a 2 normal sodium hydroxidesolution was added and the reflux was continued for another hour. Theethanol was distilled ofl and the residue acidified with dilutehydrochloric acid. An oil separated which soon solidified. The crudeacid was taken up in ether and extracted with a 5% sodium bicarbonatesolution. The sodium bicarbonate layer was separated and stronglyacidified (pH 2) with dilute hydrochloric acid. The precipitated acidwas collected by suction and washed with 50 ml. of cold water. Usuallyone recrystallization was enough to give product of analytical purityprovided the the starting materials are of good quality.

In the table below are listed numerous examples of aaryloxyalkanoicacids prepared by one or more of these methods.

Compound Method M.P. or B.P. in 0. Yield, Reference percent CH B 118-11941 Harvill, Zimmerman, Hitchcock; Contrib. I Boyce Thompson Inst. 13,273- (1944);

6.11. 48, 40098. -t I C a (3H B 136-138 37 Synerholm and Zimmerman;Contrib. Boyce, Thompson Inst., 14, 91-103 (1945);

C. A. 40, 14743. C1O-CHC 02H (g C H B 176-177 67 CH3O ONE-Q O(iJHC 02H(3H2 C HC H: B CH:

OF B (101-105/0. 15 mm.)

I @042 no OH O Ha . B 114. 5-116 31 Q-m-ona 02H 0 C H2 0 112 C H3 D72-73 25 Q-o-o HC 0 2H C H 0 a) z II c rno-Q-o- 0 -116 46 Cl O(]3HO 02H1 H; C HzC 6H5 O C,HC 0 0 148-149 40 Fawett, Osborne, Dam and Walker;Ann. Appl. Biol., 40, 231-43 (1953) cf. C.A. 45,

wherein the R groups contain only the elements carbon and hydrogen.

The following examples will serve to illustrate this invention withoutlimiting it thereto.

Compound Method M.P. or 13.1. in C. Yield, Reference percent C 125.5-126 52 S O(I7HC 211 A 125-126 63 0 Hz (1313:; C (170-173/1-15 mm.) 33cmomo-Q-o-ono one C H3 C H:

C, D H. Sobotka and J. Austin; J. Amer. Chem. HO- -O(1HC 0211 S00. 74,3813, (1952).

(31 ('31 C 173. 5-174. 5 60 o1-o-ono 0211 l I C H3 Cl Cl O-CH C(156158/0.3 mm.) 36

4 O-(l7 HC 0 1H C Ha O-CH3 Cl C 145-146 61 o6H.-o-or10 0211 CH;(\)H(CH3)2 C (129132/0. mm.) 38

| C H: CH: H3) 2 C 19 0zN- -O(IJHCO2H B 21 0 F C 90-92 21 V A 90-91. 560 O:N-O-(JHC 02H A 116-116. 5 80 A. Fredga and M. Matell Arkiv. Kemi. 3O-(|3HC 02H (nr. 325 (1951).

C H3 7 I I A 115-116 54 Fawett, Osborne, Wain and Walker; Ann. 01- O-CHC02H ApplixBiolAO, 231-43, (1953).

A 82-83 69 Fawett, Osborne, Wain and Walker; Ann. O--(| HC 01H Appll.Biol. 40, 231-43, (1953).

B 92-93 63 H. Sobotka and J. Austin; J. Amer. Chem. C 1130- -O-(JHC 02HSoc. 74, 3813 (1953).

EXAMPLE 1 Triethylamine (1.5 ml.) was added to a cold solution (10 C.)of an a-phenoxypropionic acid (1.66 gm., 0.01 mole) in 15 ml. puredioxane. The resulting clear solution was stirred and cooled to 5l0 C.while isobutyl chloroformate (1.36 gm., 0.01 mole) in 5 ml. dioxane wasadded dropwise. When the addition had .been completed the mixture wasstirred at 5-8 C. during ten minutes and then a solution of6-aminopenicillanic acid (2.16 gm., 0.01 mole) in 15 ml. Water and 2 ml.triethylamine was added dropwise while the temperature was maintainedbelow 10 C. The resulting mixture was stirred in the cold during 15minutes and then at room temperature for minutes, diluted with 30 ml.cold water and extracted with ether which was discarded. The coldaqueous solution was then covered with ml. ether and acidified to pH 2with 5 NH SO After shaking, the ether layer containing the product,6-(2-phenoxypropionamido)penicillanic acid, was dried for ten minutesover anhydrous Na SO and filtered. Addition of 6 ml. of dry n-butanolcontaining 0.373 gm./ ml. potassium 2ethylhexanoate precipitated thepotassium salt of the product as a colorless oil which crystallized onstirring and scratching and was collected, dried in vacuo and found toWeight 2.75 gm., to melt at 217219 C., to be very soluble in water, tocontain the ,B-lactam structure as shown by infrared analysis and toinhibit Staph. aureas Smith at a concentration of 0.07 mcg./ml.

EXAMPLE 2 w(2,4-dichlorophenoxy)propionic acid (0.01 mole),triethylamine (0.011 mole) and isobutyl chloroforrnate (0.01 mole) arestirred in 20 ml. pure, dry dioxane and 2 ml. dry acetone for aboutthirty minutes at about 4 C. To this solution there is then added achilled solution of G-arninopenicillanic acid (0.01 mole) andtriethylamine (0.01 mole) in 20 ml. water and the mixture is stirredabout an hour in the cold. After the addition of 1.0 gm. NaHCO in 30 ml.cold Water, the solution is extracted twice with 75 ml. portions ofether and the ethereal extract is discarded. The aqueous solution iscooled and stirred in an ice bath, covered with 75 ml. ether and adjusted to pH 2 with 5 N H 50 The ether is separated and the aqueoussolution is again extracted with 75 ml. ether. The combined etherealextracts containing the product, 6- t- 2,4-dichlorophenoxy propionamidopencillanic acid, are dried rapidly over anhydrous Na SO and filtered.The addition of 6 ml. of dry n-butanol containing 0.373 gm./ml.potassium Z-ethylhexanoate followed by additional dry ether precipitatesthe potassium salt of the product. After trituration with ether, thispotassium salt of the product is dried in vacuo over P 0 and recoveredas a water-soluble powder which inhibits the growth of Staph. aureusSmith at a concentration of 0.001 percent by weight.

EXAMPLE 3 a-(2,4-diisoamylphenoxy)-n-butyric acid (0.02 mole),triethylarnine (0.021 mole) and isobutyl chloroformate (0.02 mole) arestirred in 40 ml. pure, dry dioxane and 40 ml. dry acetone for aboutthirty minutes at about 4 C. To this solution there is then added achilled solution of -aminopenicillanic acid (0.02 mole) andtriethylamine (0.02 mole) in 40 ml. water and the mixture is stirredabout an hour in the cold. After the addition of 2.0 gm. NaHCO in 60 ml.cold water, the solution is extracted twice with 150 ml. portions ofether and the ethereal extract is discarded. The aqueous solution iscooled and stirred in an ice-bath, covered with 150 ml. ether andadjusted to pH 2 with 5 N H 50 The ether is separated and the aqueoussolution is again extracted with 150 ml. ether. The combined etherealextracts containing the product, 6-{a (2,4diisoamylphenoxy)-n-butyramido]pencillanic acid, are dried rapidly overanhydrous Na SO and filtered. The addition of 12 ml. of dry n-butanolcontaining 0.373 gm./ml. potassium Z-ethylhexanoate followed byadditional dry ether precipitates the potassium salt of the product.After trituration with ether, this potassium salt of the product isdried in vacuo over P 9 and recovered as a water-soluble powder whichinhibits the growth of Staph. aureus Smith at a concentration of 0.001percent by weight.

EXAMPLE 4 w(2,4-dichlorophenoxy)-n-butyric acid (0.04 mole),triethylamine (0.044 mole) and isobutyl chloroformate (0.04 mole) arestirred in 80 ml. pure, dry dioxane and 8 ml. dry acetone for aboutthirty minutes at about 4 C. To this solution there is then added achilled solution of 6-aminopenicillanic acid (0.04 mole) andtriethylamine (0.04 mole) in 80 ml. water and the mixture is stirredabout an hour in the cold. After the addition of 4.0 gm. NaHCO in 120ml. cold water, the solution is extracted twice with 300 ml. portions ofether and the ethereal extract is discarded. The aqueous solution iscooled and stirred in an ice bath, covered with 300 ml. ether andadjusted to pH 2 with 5 N H 80 The ether is separated and the aqueoussolution is again extracted with 300 ml. ether. The combined etherealextracts containing the product, '6- a- 2,4-dichlorophenoxy)-n-butyramido] penicillanic acid, are dried rapidly over anhydrous Na SOand filtered. The addition of 24 ml. of dry n-butanol containing 0.373gm./ml. potassium Z-ethylhexanoate followed by additional dry etherprecipitates the potassium salt of the product. After trituration withether, this potassium salt of the product is dried in vacuo over P 0 andrecovered as a water-soluble powder which inhibits the growth of Staph.aureus Smith at a concentration of 0.001 percent by weight.

EXAMPLE 5 oz (4 trifluoromethylphenoxy)-n-butyric acid (0.01 mole),triethylarnine (0.011 mole) and isobutyl chloroformate (0.01 mole) arestirred in 20 ml. pure, dry dioxane and 2 ml. dry acetone for aboutthirty minutes at about 4 C. To this solution there is then added achilled solution of 6-aminopenicillanic acid (0.01 mole) andtriethylamine (0.01 mole) in 20 ml. water and the mixture is stirredabout an hour in the cold. After the addition of 1.0 gm. NaHCO in 30 ml.cold water, the solution is extracted twice with ml. portions of etherand the ethereal extract is discarded. The aqueous solution is cooledand stirred in an ice-bath, covered with 75 ml. ether and adjusted to pH2 with 5 N H The ether is separated and the aqueous solution is againextracted with 75 ml. ether. The combined ethereal extracts containingthe product, 6-[a-(4-trifluoromethylphenoxy)-nbutyramidojlpenicillanicacid, are dried rapidly over anhydrous Na SO and filtered. The additionof 6 ml. of dry n-butanol containing 0.373 'gm./rnl. potassium 2-ethylhexanoate followed by additional dry ether precipitates thepotassium salt of the product. After trituration with ether, thispotassium salt of the product is dried in vacuo over P 0 and recoveredas a water-soluble powder which inhibits the growth of Staph. aureasSmith at a concentration of 0.001 percent by Weight.

EXAMPLE 6 a-(4-benzylphenoxy)propionic acid (0.02 mole), triethylamine(0.022 mole) and isobutyl chloroformate (0.02 mole) are stirred in 40ml. pure, dry dioxane and 4 ml. dry acetone for about thirty minutes atabout 4 C. To this solution there is then added a chilled solution of6-aminopenicillanic acid (0.02 mole) and triethylamine (0.02 mole) in 40ml. water and the mixture is stirred about an hour in the cold. Afterthe addition of 2.0 gm. NaHCO in 60 ml. cold water, the solution isextracted twice with ml. portions of ether and the ethereal extract isdiscarded. The aqueous solution is cooled and stirred in an ice-bath,covered with 150 ml. ether and adjusted to pH 2 with 5 N H 80 The etheris separated and the aqueous solution is again extracted with 150 ml.ether. The combined ethereal extracts containing the product, 6 [a(4-benzylphenoxy)propionamido] penicillanic acid, are dried rapidly overanhydrous Na SO and filtered. The addition of 12 ml. of dry nbutanolcontaining 0.373 gm./ml. potassium 2-ethylhexanoate followed byadditional dry ether precipitates the potassium salt of the product.After trituration with ether, this potassium salt of the product isdried in vacuo over P 0 and recovered as a Water-soluble powder whichinhibits the growth of Staph. aureus Smith at a concentration of 0.001percent by weight.

EXAMPLE 7 In the procedure of Example 1, the a-phenoxypropionic acid isreplaced by 0.10 mole a-(2-chlorophenoxy)propionic acid,a-(p-sulfamylphenoxy)-n-butyric acid, a-

1 1 (3,4-dimethoxyphenoxy)-n-pentanoic acid,a-(3-methylphenoxy)-iso-valeric acid, u-(4-dimethylaminophenoxy)-n-hexanoic acid, a-(2-methoxyphenoxy)-n-decanoic acid, on(2,4-dichlorophenoxy)phenylacetic acid, a-(2-nitrophenoxy) [3phenylpropionic acid, a-(Z-acetamidophenoxy)-'y-pheny-lbutyric acid,a-(2,4-dimethylphenoxy)-nbutyricacid, a-(4-isopropylphenoxy)propionicacid, u-(S- bromophenoxy)-n-butyric acid, a-(2-iodophenoxy) phenylaceticacid, a-(2-ethylaminophenoxy)- iso-valeric acid, a (2,5dihydroxyphenoxy)-iso-hexanoic acid, u-(4-hydroxyphenoxy)propionic acid,a-phenoxy-iso-valeric acid, u-phenoxy-n-decanoic acid,a-phenoxy-y-phenylbutyric acid, a-(2-benzylphenoxy)-n-butyric acid,oz-(Z-tliflllOl'O- methylphenoxy)propionic acid, and a-(4-aminophenoxy)propionic acid, respectively, to produce the acids 6- [a-(2-chlorophenoxy)propionamido1penicillanic acid, 6- a-(4-sulfamylphenoxy -n-butyramido] pencillanic acid, 6[a-(3,4-dimethoxyphenoxy) -n-pentanoamido] pencillanic acid, 6- oc-S-methylphenoxy -iso-valeramido] pencillanic acid, 6-[w(4-dimethylaminophenoxy)-n-hexanoamido] penicillanic acid,6-[a-(2-methoxyphenoxy)-n-decanoamido] penicillanic acid, 6- a-(2,4-dichlorophenoxy phenylacetamido] penicillanic acid,6-[a-(2-nitrophenoxy)-p-phenylpropionamido] penicillanic acid,6-[u-(2-acetamidophenoxy)-'y-phenylbutyramido] penicillanic acid,6-[u-(2,4-dimethylphenoxy)-n-butyramid0] penicillanic acid,6-[a-(4-isopropylphenoxy)propionamido1penicillanic acid, 6- oc-3-bromophenoxy) -n-buty-ramido] penicillanic acid, 6- [a- 2-iodophenoxy)phenylacetamido] penicillanic acid, 6- [a- 2-ethylaminophenoxy)-iso-valeramido] penicillanic acid,6-[a-(2,5-dihydroxyphenoxy)-iso-hexanoamido] penicillanic acid, 6- a-(4-hydroxyphenoxy) propionamido] penicillanic acid, 6-u-phenoxy-iso-valeramido] penicillanic acid, 6- u-phenoxy-n-decanoamido] penicillanic acid, 6- [a-phenoxy-vphenylbutyramido] penicillanicacid, 6- a- (2-benzylphenoxy -n-butyramido'] penicillanic acid, 6- [oc-Z-trifluoromethylphenoxy) propionamido] penicillanic acid, and 6-[a-(4-aminophenoxy)propionamido] penicillanic acid, respectively, whichare isolated as their solid, watersoluble potassium salts and found toinhibit Staph. aureus Smith at concentrations of 0.001 percent byweight.

EXAMPLE 8 To a cooled, stirred solution of a-phenoxyphenylacetic acid(6.8 g., 0.03 mole) in 40 m1. pure dry dioxane there was added dropwise4.2 ml. (3.0 g., 0.03 mole) triethylamine (4.2 ml.) in 25 ml. water. Thereaction mixture isobutyl chloroformate (4. 1 ml., 4.1 g., 0.03 mole).After twice extracted into 100 ml. ether. The combined ethereal extractscontaining the product, 6-(a-phenoxyphenylacetamido)pencillanic acid,were Washed once with cold water, dried quickly over anhydrous sodiumsulfate and filtered. The addition of 15 ml. of dry n-butanol containing0.373 g./ml. potassium 2-ethylhexanoate precipitated the potassium saltof the. product as a gum which was dissolved in methyl isobutyl ketone.Addition of about twenty volumes of dry ether precipitated this salt asan amorphous white solid which was collected, dried in vacuo over P andfound to weigh 11.0 g., M.P. 88- 95. C., decomposed at 120-125 C., tocontain the B- lactam ring as shown by infrared analysis and to inhibitStaph. aureus Smith at a concentration of 0.2 mcg./ml.

EXAMPLE 9 a-(p-Tert-butylphenoxy)propionic acid (0.03 mole, 6.68 g.),triethylamine (4.22 ml., 0.03 mole) and isobutyl chloroformate (3.9 ml.,0.03 mole) were dissolved at 0-3 C. in 40 ml. pure, dry dioxane and 5ml. acetone and the solution was stirred twenty minutes. There was thenadded a solution chilled to 03 C. of 6-aminopenicillanic acid (6.5 -g.,0.03 mole) and triethylamine (4.22 ml., 0.03 mole) in 40 ml. water andthe reaction mixture was stirred for one hour. The mixture was adjustedto pH 8 with aqueous NaHCO and extracted with ether, which wasdiscarded. The aqueous phase was covered with ether, acidified with 6 NHCl to pH 2 and twice extracted with ether. The combined etherealextracts containing the product, 6-[a-(p-tert.-butylphenoxy)propionamido]penicillanic acid, were washed with cold water, dried overanhydrous Na SO and filtered. The addition of 5 ml. ml. would have beenpreferable) of potassium Z-ethylhexanoate converted the product to itspotassium salt which was precipitated as a white, crystalline solid upondilution with anhydrous ether, collected by filtration, dried overnightin vacuo over P 0 and found to weight 6.2 g., to melt at 219-220" C.with decomposition, to contain the fl-lactam ring as shown by infrared"analysis and to inhibit Staph. aureus Smith at a concentration of 0.1mcg./ ml.

EXAMPLE 10 The procedure of Example 9 was followed except that thea-(p-tert-butylphenoxy)propionic acid was replaced by2-(p-tert.-amylphenoxy)-n-butyric acid (7.53 g., 0.03 ml., also calleda-[p-(1,1-dimethylpropyl) phenoxy]-nbutyric acid) to produce the finalproduct, potassium 6- [a-(p-tert-amylphenoxy)-n butyramido]penicillanateas a solid gum (after trituration with ether) which was converted to afine powder on drying in a vacuum desiccator over P 0 This salt weighed4.7 g., on heating slowly decomposed above C., contained the fl-lactamring as shown by infrared analysis and was found to inhibit Staph.aureus Smith at a concentration of less than 10 meg/ml.

EXAMPLE l1 6-(2-phenoxypropionamido)penicillanic acid was alsoconveniently prepared by direct acylation of fermentation broth (beer)followed by isolation therefrom of the penicillin as its potassium salt.Thus, a submerged aerobic fermentation of Penicillium chrysogenum wascarried out according to the general methods for the production ofpenicillin G except for the omission of the usual addition ofphenylacetic acid as the precursor. At the end of the fermentation thebroth was filtered and its pH was adjusted to 7.5 with 10% sodiumhydroxide. After cooling the filtered broth to about 10-20 C. there wasadded with agitation 5 moles of a-phenoxypropionyl chloride to thefiltered broth for each mole of 6-aminopenicillanic acid therein asdetermined by analysis. The phenoxypropionyl chloride was added as a 5%solution in acetone, i.e. approximately .00031 moles of acid chlorideper ml. The pH dropped during the addition of a-phenoxypropionylchloride and, therefore, the rate of addition was kept slow enough toenable the pH to be maintained at 7.5 by the addition of 10% sodiumhydroxide. The reaction mixture was then agitated at 1020 C. for anadditional 30 minutes. The reaction was presumed to be complete when thepH remained constant; this required about 10-15 minutes. To avoidcontamination with acid-labile penicillins present in the broth, thesewere destroyed by next lowering the pH of the reaction mixture to pH 2for 30 minutes before subsequent solvent extraction (the same resultcould also be obtained by originally filter ing the broth at pH 2 andholding it there for 30 minutes). The acidified reaction mixture wasextracted with /2 volume of methyl isobutyl ketone for 20 minutes andthe methyl isobutyl ketone containing the desired 6-(2-phenoxypropionamido)penicillanic acid was separated and filtered toobtain sparkling solvent free of a separate water phase. This acidpenicillin in the solvent was then converted to its potassium salt bystirring the methyl isobutyl ketone solution vigorously with 5% byvolume of aqueous potassium acetate buifer having a specific gravity of1.30 while cooling to 510 C. Crystallization of the product, potassium6-(Z-phenoxypropionamido) penicillanate started almost immediately andafter one hour the crystalline product was collected by filtration,washed successively with methyl isobutyl ketone, dry butanol and acetoneand found to be a potent, white crystalline solid.

In numerous runs by this procedure the efiiciency of the activation stepwas generally about 80% with the remaining 6-aminopenicillanic acidaccounted for in the spent broth. From activated broth to crudepenicillin the activity yield was 77%. On a molar basis 0.64 mole of thenew penicillin was produced per mole of 6-aminopenicillanic acid in thebroth. Thus, in one series of runs 11.9 kg. of crude penicillin wasproduced from 5500 gallons of broth.

EXAMPLE 12 To a-(2,4-dichlorophenoxy)propionic acid (6.53 g., 0.0278 m.)dissolved in 12 ml. acetone and 60 ml. dioxane there was added 4.0 cc.(0.0280 ml.) dry triethylamine. The solution Was cooled to C. and therewas added 3.64 cc. (0.0278 in.) isobutyl chloroformate and the mixturewas stirred for 20 minutes. To this solution there was added at C. asolution of 6.00 g. (0.0278 In.) of 6-aminopenicillanic acid in 60 ml.water and 4.0 cc. triethylarnine. Evolution of carbon dioxide was briskand the mixture was stirred for 90 minutes at 05 C. To the reactionmixture there was then added a solution of 2.0 g. sodium bicarbonate in60 ml. water cooled to C. The mixture was twice extracted with 150 cc.poitions of cold ether which were discarded. After adding 10 -cc. 6 Nhydrochloric acid with cooling, the mixture was twice extracted with 150cc. cold ether. The combined ethereal extracts containing the product,6(2,4-dichlorophenoxy-Z-propionamido)penicillanic acid were washed with50 cc. cold water, dried over anhydrous sodium sulfate and filtered. Theaddition of cc. of dry n-buta- 1101 containing 0.373 gm./ml. potassium2-ethylhexanoate' precipitated the potassium salt of the product as awhite solid which was collected by filtration, dried in vacuo over P 0and found to weigh 8.00 g., MP. 203-205 C. with dec., to contain theB-lactam ring as shown by infrared analysis and to inhibit Staph. aureusSmith at a concentration of 0.2 mcg./ml., and to exhibit versus Staph.aureus Smith upon intramuscular injection in mice a CD of 3 mgm./kg.

Analysis.Calcd for C17H17Cl N2O5SK: C, H, 3.69; N, 5.94. Found: C,41.73; H, 3.77; N, 5.10.

there was added 105 g. (1.25 mole) sodium bicarbonate.

After stirring for one hour in an ice bath there was added 54 g. (0.25mole) 6-aminopenicillanic acid. This slurry was stirred for thirtyminutes in an ice bath and then there was added dropwise over thirtyminutes with vigorous stirring at a maximum temperature of 10 C. asolution in 100 ml. acetone of Z-phenoxypropionyl chloride (68.8 g.,0.375 mole). Methyl isobutyl ketone (400 ml.) was added and the vigorousstirring was continued for five minutes. After separating and discardingthe methyl isobutyl ketone, the aqueous layer was extracted with 250 ml.portions of methyl isobutyl ketone which were also discarded. Theaqueous layer was cooled, acidified to pH 2 with 40% H in an ice bathand extracted with a total of 800 ml. methyl isobutyl ketone. Thecombined solvent extracts containing the product, 6-(2-phenoxypropionamido)penicillanic acid, were dried over anhydroussodium sulfate for two hours in an ice bath and filtered. The productwas then converted to its potassium salt by the addition of ml. of 50%potassium Z-e-thylhexanoate in n-butanol to precipitate 67 g. ofcrystalline potassium salt of the product.

EXAMPLE 14 Potassium 6-(2-phenoxybutyramido)penicillanate was preparedaccording to the procedure of Example 1 using 6-aminopenicillanic acid(21.6 g., 0.1 m.) in 100 ml. water and sufficient triethylamine todissolve, 2-phenoxybutyric acid (18 g., 0.1 m.) in 80 mi. p-dioxane and20 ml. pure acetone and 13.7 ml. isobutyl chloroformate. There wasobtained 10.3 g. product, M.P. 197 C. with dec. (darkened at 170 C.)which was very soluble in water, contained the B-lactam structure asshown by infrared analysis, inhibited Staph. aurezts Smith at aconcentration of 0.05 mcg./ml. and exhibited versus Staph. aureus Smithupon intramuscular injection in mice a CD of 1.1 mgm./kg.

AIZQlySiS.-C?J.1CCl for C13H21N2O5SK: C, H, Found: C, 51.1; H, 5.49.

EXAMPLE 15 Potassium 6 [ot-(2,5-dichlorophenoxy)propionamido]penicillanate was prepared according to the general procedure of Example1 using a-(2,5-dichl-orophenoxy) propionic acid (0.1 m., 23.5 g.) in 160ml. dimethylformamide and 14 ml. triethylamine, 6-aminopenicillanic acid(0.1 m., 21.6 g.) in 160 ml. water and 14 ml. triethylamine and isobutylchloroformate (0.1 m., 13.7 g.). There was obtained 21.5 g. product,M.P. ZOO-204 C. with dec. (darkens above C.) as a solid which was foundto be very soluble in water, to contain the fi-lactam structure as shownby infrared analysis, to inhibit Staph. aureus Smith at a concentrationof 0.05 mcg./ml. and exhibited versus Staph. aureus Smith uponintramuscular injection in mice a CD of 2.5 mgm./kg.

EXAMPLE 16 Potassium 6 [a-(p-acetamidophenoxy)propionamido]penicillanate was prepared according to the procedure of Example 1 using31 g., 0.15 m. a-(p-acetamidophenoxy)propionic acid and obtained as 27.7g. solid, M.P. 211-215 C. dec. when put on a rapidly heating stage at200 C. and found to be very soluble in water, to contain the fl-lactamstructure as shown by infrared analysis, to inhibit Staph. aureus Smithat a concentration of 0.4 mcg./ml. and to exhibit versus Staph.azzreusSmith upon intramuscular injection in mice 9. CD of 6.4 mgm./kg.

Analysis.Calcd for C19H22N305SK: C, H, 4.70. Found: C, 50.12; H, 5.21.

An additional yield of 15.9 g. was obtained by adding more acetone tothe mixture of methyl isobutyl ketone and 50% potassium 2-ethylhexanoatein butanol. This fraction melted at 223226 C. with dec. but some meltingwas observed starting at about 214 C.

EXAMPLE l7 Thionyl chloride (30 ml.) was added fairly rapidly at roomtemperature to a cool solution of 200 ml. benzene, 1 ml. pyridine and 21g. (0.0725 mole) u-(2-benzyl-4- chlorophenoxy)propionic acid and themixture was refluxed for two hours and then the solvents were removed bydistillation in vacuo up to 100 C. at 20 mm. The residual acid chloridewas cooled and dissolved in 80 m1. acetone and added slowly to a mixtureof 13 g. (0.06 mole) 6-arninopenicillanic acid in 100 ml. watercontaining 12.6 g. (0.15 mole) sodium bicarbonate and 20 ml. acetone.Proceeding according to Example 13, there was obtained 13.4 g. solidpotassium 6-[a-(2-benzyl-4- chlorophenoxy)propionamido]penicillanate,melting at 230231 C. with decomposition, which was soluble in water,contained the fi-l-actam structure as shown by infrared analysis,inhibited Staph. aurem' Smith at a concentration of 0.4 mcg./ml.,exhibited versus Staph. aureus Smithupon intramuscular injection in micea preliminary CD of 9 mgm./kg., and was only 26% inactivated by pH 2.5acid under conditions which decomposed benzylpenicillin to the extent ofgreater than 96%, i.e. in 0.75 molar citric acid for one hour at 37 C.

EXAMPLE 18 After dissolving a-phenoxy caproic acid (M.P. 70-72 C., 13.70g., 0.0659 m.) in 150 ml. dry dioxane and 30 ml. dry acetone and 9.28cc. (0.0659 m.) dry triethylamine and cooling to 2 C. there was added8.65 cc. (0.0659 m.) isobutyl chloroformate. The temperature rose to 6C. and the mixture was stirred at to 2 C. for 25 minutes. To this coldmixture there was then added a solution prepared by mixing 9.3 cc.triethylamine, 19.7 ml. water and 14.2 g. (0.0659 m.)6-aminopenicillanic acid at C. for five minutes. Carbon dioxide wasevolved rapidly from the stirred mixture and the formation of thetriethylamine salt of the product, 6-(a-phenoxycaproamido)penicillanicacid for 6-(a-phenoxy-n-hexanoamido) penicillanic acid, was essentiallycomplete after twenty minutes at 5-8 C. After stirring for an additionalthirtyfive minutes there was added a cold solution of 9.8 g. NaHCO in260 ml. water and the mixture was twice extracted with 200 ml. coldether which was discarded. The mixture was then acidified with 37 ml.cold 6 N H 80, and twice extracted with 200 ml. cold methyl isobutylketone. The combined solvent extracts containing the product,6-(ot-phenoxycaproamido)penicillanic acid, were washed with m1. icewater, dried over Na SO and filtered. The addition of 27 ml. of 50%potassium Z-ethylhexanoate in dry n-butanol converted the product to itspotassium salt which was precipitated as a mobile oil after successiveadditions of 500 ml. dry ether and 1000 ml. lower alkanes (SkellysolveB). The product was recovered by decantati-on, washed four times with400 m1. dry ether and dried in vacuo to give 22.2 g. of amorphous solidproduct as the potassium salt. This salt was very soluble in water,decomposed on heating at about 160 C., contained the fl-lactarnstructure as shown by infrared analysis, inhibited Staph. aarezts Smithat a concentration of 0.4 mcg./ml., exhibited versus Staph. aureus Smithupon intramuscular injection in mice a preliminary CD of 4 mgin./kg. andwas only 36% inactivated by pH 2.5 acid under conditions whichdecomposed benzylpenicillin to the extent of greater than 96% i.e. in0.75 molar aqueous citric acid for one hour at 37 C.

EXAMPLE 19 a-(4-chloro-3,S-dimethylphenoxy)caproic acid (0.65 m., 17.6g.) was used in the procedure of Example 18 to produce 6-[a-(4-chloro-3,S-dimethylphenoxy)caproamido]penicillanic acid which wasisolated as its potassium salt in the form of a fluffy, amorphous,hygroscopic solid which weighed 10.0 g., melted at 105108 C. withdecomposition, was soluble in water, contained the ,3- lactam structureas shown by infrared analysis, inhibited Staph. aureus Smith at aconcentration of 0.4 meg/ml, exhibited versus Stap. aztreus Smith uponintramuscular injection in mice a preliminary CD of 9 mgm./kg., was only29% inactivated by 1 u./ ml. penicillinase under con;

15 ditions which inactivated benzylpenicillin to the extent of 73% andwas only 55% inactivated by pH 2.5 acid under conditions whichdecomposed benzylmnicillin to the extent of greater than 96%, i.e. in0.75 'molar aqueous citric acid for one hour at 37 C.

EXAMPLE 20 a-Phenoxy-n-valeric acid (0.1 m., 19.4 ml.) was used in theprocedure of Example 18 on a 0.1 mole scale and methyl isobutyl ketonewas used as the extraction solvent to produce 6 (c:phenoxycaproamido)penicillanic acid which was isolated as its potassiumsalt in the form of solid weighing 17.7 g., melting at 170-175 C. with.

EXAMPLE 21 Potassium 6 [a (2,4,6trichlorophenoxy)propionamido1-penicillanate was prepared froma-(2,4,6-trichlorophenoxy)-propionic acid (0.1 m., 27 g.) according tothe procedure of Example 15 and recovered as a crystalline solid, 4.9g., M.P. 163165 C. with decomposition (darkens slowly above C.). Theproduct was soluble in water, contained the fl-lactam structure as shownby infrared analysis, inhibited Staph. aurezts Smith at a concentrationof 0.4 mcg./ml., exhibited versus Staph. aureus Smith upon intramuscularinjection in mice a preliminary CD of 9 mgm./kg., was only 37%inactivated by 1 u./ml. penicillinase under conditions which inactivatedbenzylpenicillin to the extent of 58% and was only 27% inactivated by pH2.5 acid under conditions which decomposed benzylpenicillin to theextent of greater than 96%, i.e. in 0.75 molar aqueous citric acid forone hour at, 37 C.

Analysia-Calcd for C H -Cl KN2O S: C, 40.3; H, 3.19. Found: C, 40.8; H,3.55.

EXAMPLE 22 Potassium 6-[ot (p methoxyphenoxy)propionamido] penicillanatewas prepared from 19.6 g. (0.1 mole) a- (p-methoxyphenoxy)-propionicacid according to the procedure of Example 15 and recovered as a whitecrystalline solid, 14.6 g., M.P. 211214 C. with decomposition withdarkening above 208 C. The product was soluble in water,.contained theB-lactam structure as shown by infrared analysis, inhibited Staph.aureus Smith at a concentration of 0.1 mcg./ml., exhibited versus Staph.aztreus Smith upon intramuscular injection in mice a preliminary CD of1.9 mgm./kg., was only 23% inactivated by 1 u./ml. penicillinase underconditions which inactivated benzylpenicillin to the extent of 5 8% andwas only 2% inactivated by pH 2.5 acid under conditions which decomposedbenzylpenicillin to the extent of greater than 96%, i.e. in 0.75 molarcitric acid for one hour at 37 C.

Analysis.Calcd for C H KN O S: C, 49.9; H, 4.90. Found: C, 49.66; H,5.10.

EXAMPLE 23 a (m Trifluoromethylphenoxy)propionic acid (0.1 m., 23.4 g)was used in the procedure of Example 18 on a 0.1 mole scale to produce6-[a-(3-trifluoromethylphenoxy)propionamido]-penicillanic acid which wasisolated as its potassium salt in the form of a white solid whichweighed 11.0 g., melted at 188190 C. with decomposition, contained theti-lactam structure as shown by infrared analysis, inhibited Staph.aureus Smith at a con- 1 7 centration of 0.1 meg/ml. and exhibitedversus Staph. aureus Smith upon intramuscular injection in mice apreliminary CD of 1.8 mgm./kg.

EXAMPLE 24 a-(p-Nitrophenoxy)propionic acid (0.1 m., 22.1 g.) was usedin the procedure of Example 18 on a 0.1 mole scale to produce 6-[oc (4nitrophenoxy)propionamido]penicillanic acid which was isolated as itspotassium salt in the form of a solid which weighed 31.8 g., melted at202203 C. with decomposition, contained the fi-lactam structure as shownby infrared analysis and inhibited Staph. aureus Smith at aconcentration of 0.8 meg/ml.

EXAMPLE 25 EXAMPLE 26 The potassium salts of 6 [c (2,4dibromophenoxy)propionamido]penicillanic acid, 6 [a(Z-n-pentylphenoxy)propionamido]penicillanic acid, '6 [a (2,6-dimethoxyphenoxy)propionamido] penicillanic acid, 6- [a (2,3,4,5,6pentachlorophenoxy)propionamido]penicillanic acid and 6 [cc (4 nitro 3trifluoromethylphenoxy)propionamido]penicillanic acid were preparedaccording to the methods described above, isolated as colorless solidsand found to be soluble in water and to inhibit Staph. aureus Smith atthe following concentrations in meg/ml. respectively: 0.05, 0.4, 3.1,0.1 and 0.2.

EXAMPLE 27 Potassium 6 [a (pcyclohexylphenoxy)propionamido]penicillanate was prepared from a (pcyclohexylp'henoxy)propionic acid (30 g., 0.121 mole) according to theprocedure of Example 17 and found to weigh 37 g., to melt at 23023l C.with decomposition, to contain the ,B-lactam structure as shown byinfrared analysis and to inhibit Staph. aureus Smith at a concentrationof 0.4 meg/ml.

EXAMPLE 28 Potassium 6-[a-(3,5-dimethylphenoxy)propionamido]-penicillanate was prepared according to Example 18 from 19.4 g. (0.1mole) a-(3,S-dimethylphenoxy)propionic acid and recovered as a whitecrystalline material, 21.5 g., M.P. 220-222 C., which containedtheli-lactam structure as shown by infrared analysis and inhibitedStaph. aureus 7 Smith at a concentration of 0.1 meg/ml.

EXAMPLE 29 The two diastereoisomers of6-(a-phenoxypropionamido)penicillanic acid were prepared in the form of their potassium salts byresolving racemic a-phenoxypropionic acid using yohimbine, convertingthe dextro and levo isomers of the acid to the corresponding acidchlorides and then reacting each isomer with 6-aminopenicillanic acidobtained from a fermentation broth. This resolution was accomplishedfrom the method of E. Fourneau and G. Sandulesco, Bull. Soc., Chim.,Ser. 4, 31,988-990 (1922), with some attention to the work of A. Fredgaand M. Matell, Arkiv. Kemi, 4, 325-330 (1952). The final products werearbitrarily named as the alpha and beta or D- and L-isomers.

Resolution of dl u-phenoxy'propionic acid using yohimbine (A) Isolationof d-a-phenoxypropionic acid-To a mixture of 170 grams (0.435 mole) ofyohimbine hydrochloride in 8.7 liters of water was added 200 ml. ofammonium hydroxide. The mixture was stirred for one hour and filtered.The yohimbine base was washed with distilled water, dried as well aspossible on the filter, and transferred into 2.03 liters of hot ethanol(ca. and 8.7 liters of water. A total of 96.3 grams (.58 mole) of dla-phenoxypropionic acid was added, the mixture was brought up to ca. andnearly all the solids dissolved. The solution was filtered to remove asmall amount of gummy material present as an impurity in the originalacid. The filtrate was stored in the cold room overnight. In the morningdense crystals had deposited (it was found that heating of the solutionon the steam bath speeded up the crystallization before storage). Thesalt was filtered and weighed 132 grams (crude). The filtrate was calledFilt-rate A. The yohimbine d-(u-phenoxypropionate) was recrystallized bydissolving in 1 liter of absolute alcohol with heating on the steam bathand diluting with 8 liters of water. After storage overnight in the coldroom the solid was filtered and weighed about 75 grams, M.P. 207.5- 212C., [a] +40.74 (c., 1 alcohol). The wet sample was slurried with 200 ml.of 5% Na CO for one half hour. The mixture was filtered and theyohimbine was dried in air for three days; weight 39.5 g. The sodiumcarbonate solution was layered with 200 ml. of ether and concentratedhydrochloric acid was added until the pH was 2. The two layers wereseparated after vigorous shaking and the organic layer was washed withwater and dried over anhydrous sodium sulfate. The ether was evaporatedand 12 grams of d a-phenoxypropionic acid was obtained, M.P. 86.5-88 C.,[a] |39.l2 (c., 1 alcohol).

' (B) Isolation of l-ot-phenoxypropionic acid.--Filtrate A wasevaporated to dryness under reduced pressure on the steam bath to awhite solid. The solid was stirred one hour with 300 ml. of 5% Na COsolution and the yohimbine was filtered off and weighed, after drying,88 grams. The sodium carbonate solution was layered with 200 ml. ofether and acidified to pH 2 with concentrated hydrochloric acid. Theether layer was stirred vigorously, washed with water and dried overanhydrous sodium sulfate. Upon evaporation a white solid was obtainedM.P. 111-115" C. [a] -8.21 (c., 1 alcohol). The acid weighed 20 grams.The solid was partially dissolved in 200 ml. of hot cyclohexane andfiltered to give 10 g. of racemic dl a-phenoxypropionic acid M.P.ll5-ll6 C., [od 0 (c., 1 alcohol). The cyclohexane was evaporated todrymess to give 5 grams of primarily l-a-phenoxypropionic acid M.P.86-87? [q] -36.5. The sample was recrystallized from 100 ml. of hotcyclohexane to give 3 grams of the levo enantiomorph, M.P. 87-88 C., M137.8 (c., 1 alcohol).

(C) Preparation of potassium 6-(D-u-phenoxypropi0namido)penicillanate(alpha isomer).The general procedure of Example 1 was followed using 8.3g. (0.05 m.) of dextro-a-phenoxypropionic acid in 40 ml. p-dioxane and20 ml. pure acetone, 10.8 g. (0.05 m.) 6-aminopenicillanic acid in 60ml. water and 10 ml. triethylamine, 7 ml. isobutyl chloroformate and 10ml. triethylamine to give 7.2 g. of the solid potassium salt, [a] +225,c.=l in water, M.P. 219-220 C. with decomposition.

This isomer was also called the alpha isomer or potassiumD-a-phenoxyethylpenicillin. The alpha isomer Was found to exhibit versusStaph. aureus Smith upon intramuscular injection in mice a CD of 1.2mgm./ kg.

(D) Preparation of potassium 6-(L-a-phenoxypropionamido) penicillanate(beta isomer).The above procedure was followed using 2 g. (0.012 m.) ofIBVO-ot-PhCIlOXY- propionic acid in .10 ml. p-dioxane and 5 ml. pureacetone, 2.6 g. (0.012 m.) 6-aminopenicillanic acid in 15 ml. water 19and 2 ml. triethylamine, 1.7 ml. (0.0121 111.) isobutyl chloroformateand 2 ml. triethylarnine to give 4.6 g. of the potassium salt, [a] +204,c.=1 in water, M.P. 221- 222 C. with decomposition.

This isomer was also called the beta isomer of potassiumL-a-phenoxyethylpenicillin. The beta isomer was found to exhibit versusStaph. aureus Smith upon intramuscular injection "in mice a CD of 0.6mgm./kg.

A mixture of equal parts by weight of these alpha and beta isomers wasfound to exhibit versus Staph. aareus Smith upon intramuscular injectionin ten mice a CD of 0.45 mgm./kg. In other experiments it was repeatedlynoted that a mixture of these isomers, whether prepared as above or byacylation of 6-a-minopenicillanic acid with racemic u-phenoxypropionylchloride, gave either a lower CD than either pure isomer or a CDsubstantially equal to that give by the more potent (beta) isomer.

Samples of potassium 6-(2-phenoxypropionamido)penicillanate prepared asin Example 111 above by acylation of broth containing6-aminopenicillanic acid by the use of racemic a-phenoxypropionylchloride were found by infrared analysis (in potassium bromide pelletsat 13.08 and 13.18 microns) and phase solubility analysis in 95% ethanolto contain as their major element an amount of 60 to 80% :5% by weightof the beta (levo) isomer. The pure beta isomer had a solubility in 95%ethanol at room temperature of 5.5-6.0 mgm./ g. of solvent and could beobtained from the mixture by successive leechings of the more solublealpha isomer by 95% ethanol. Pure samples of the beta isomer were alsoobtained by dissolving in warm 95 ethanol enough of the mixture ofisomers to provide slightly more than 6 mgm. beta isomer per gram ofsolvent and then allowing the mixture to cool to room temperature.

Alpha D isomer d-a-Phenoxypropionic acid (83.31 g., 0.5 mole, [a] =+40,c.=1, C H OH) in 500 ml. pure dioxane and 200 ml. acetone was converted,by reaction in the usual manner with 70 g. isobutyl chloroformate and 75ml. triethylamine and 108 g. (0.5 mole) 6-aminopenicillanic acid in 700ml. water and sufiicient triethylamine to dissolve, to the alpha isomer,potassium 6-(D-2-phenoxypropionamido)penicillanate, 134 g., [a] +250 (1%in water), M.P. 240241 C. with decomposition. Recrystallization of 100g. from -1 liter n-butanol and 200 ml. water. gave 61 g. material having[a] +251, 1% in water, and decomposing on heating at 236237 C.

By the same procedure on a 0.026 mole scale, levo a-phenoxypropionicacid (4.3 g., [a] 39.5, c.=1 in C H OH) was converted to the betaisomer, potassium 6-(L-Z-phenoxypropionamido) penicillanate, andrecrystallized (6 g. from 100 ml. n-butanol and 20 ml. water) to give4.75 salt exhibiting [eth l-218, 1% in water.

The beta (L) isomer could also be isolated in pure form from solidsprepared by broth acylation in which it was present in a ratioof :30 byrepeated recrystallizations from n-butanol-water. Thus, 500 g. wasrecrystallized from three liters n-butanol and 900 ml. water to give 200g. ([oz] +218, 1% in water, decomposition point 242-243 C.). A secondrecrystallization gave 125 g. [a] +218221, 1% in water, decompositionpoint 245-246 C.

Analysis.Calcd for C H 9N O SK: C, 50.73; H, 4.76;N, 6.96. Found: C,50.65; H, 4.83; N, 6.82; H O, none. A third recrystallization gave 73 g.([a] +220, 1% in water; decomposition point 239240 C.).

Microbiological studies on BL-PI52 Studies were done on the [a (D) and B(L)] diastereoisomers obtained from the reactions of D- andL-a-phenoxypropionic acid with 6-aminopenicillanic acid as well as on amixture of the two diastereoisomers representing a typical lot producedby broth acylation with racemic acid chloride. The separateddiastereoisomers will be designated as alpha isomer" and beta isomerrespectively, and the mixture of isomers will be designated as mixture.Comparisons of these materials were made with penicillin V..All were inthe form of potassium salts.

Spectrum of BL-P152 The minimum inhibitory concentration (MIC) wasdetermined for a variety of microorganisms using serial dilutiontechniques. The inoculum was a 10 dilution of an overnight broth cultureexcept for those cultures which grewpoorly when a 10 dilution was used.The growth was read after eighteen hours at 37 C. The results are listedbelow in Table 1.

TABLE A.COMPARISON OF MINIMUM INHIBI'IO RY CONCENTRATIONS IN MC GJML. IN

HEART INFUSION BROTH MIG Organism Alpha Beta Mixture Pen. V.

Isomer Isomer 1? (I'Itflllfl it 0.25 0.06 0.03 0. 03 Bacillus CCTMIQ 012.5 5 25 Bacillus circulans ATCC 9961 6.25 6.25 6. 25 3.1 Cory -tumxcrosis 0.125 0.06 0.03 0.03 Diplococcus pucumomae' 0. 06 0. 06 0. 06 0.03 Escherichia coli ATCC 8739 100 100 100 Gaffkya Tetragcua 0.03 0.0150.015 0.07 Micrococcus flaunt v 0.125 0.015 0.015 0.007 Salmonellaporatyphi A. 50 25 25 12. 5 S 7 phnm 100 100 100 100 Sarcina Zutea ATCC10054 0.12 0.007 0.007 0.007 Shigclla sonnet 100 100 100 25Staphylococcus aurcus 209P 0. 0. 06 0. 03 0. 03 Staphylococcus aureusvar. Smith 0.125 0. 03 0.03 0. 03 Streptococcus agalactiae ATCC 10770.06 0.03 0.03 0. 03 Stre s ATCC 9 0.06 0.03 0. 03 0.03 Sir p "f PCI110; 45 6.25 6. 25 25 Streptococcus pyogenes 203 0. 06 0. 06 0. 06 0.015 Streptococcus pyogenes Digonnet..- 0.015 0. 03 0.06 0.03Streptococcus py'ogencs N0. 2340 0. 06 0. 06 0.03 0.03 Streptococcuspyogenes No. 23586 0.06 0. 06 0. 06 0. 03 Vibrz'o commav 25 50 25 12. 5

Efiect on resistant staphylococcal s't'rains When MICs are determinedagainst a variety of clinically isolated resistant staphylococcalstrains, the inhibitory end points are invariably lower than thoseobtained with penicillin G or V. This is shown below in Table 2.

TABLE 2. COMPARISON F MINIMUM INHIBITORY CONCENTRATIONS USING PENICILLINRESISTANT STRAINS OF S. AUREUS OF CLINICAL ORIGIN MIC (mcgJmL) S. aureusStrain No.

Alpha Beta Mixture Pen. V. Pen. G. Isomer Isomer Effect of penicil linase The rates of inactivation of potassium6-(a-phenoxypropionamido)penicillanate (the mixture) penicillin G, andpenicillin V by Bacillus cereus penicillinase were determined. Theresults in Table 3 represent the inactivation of these penicillins byfive units of Penicillinase A, SchenLabs. It will be seen thatpotassium6-(tx-phenoxypropionamido)penicillanate (the mixture) is considerablymore resistant to B. cereus penicillinase than penicillin V or G.

In vivo mouse protection tests Comparative animal protection tests werecarried out using Staphylococcus aureus (Smith) as the infectingorganism. The antibiotics were given intramuscularly at the time ofinfection and the amount of antibiotic necessary to cure half theanimals was determined (CD The data are listed in Table 5.

TABLE 5.-CD50 VALUES OF PENICILLINS USING STAPHYLOO'OC'C'US AUREUS(SMITH) Penicillin: CD (mg/kg.) Alpha isomer 0.85 Beta isomer 0.35Mixture 0.18 Penicillin V 0.64 Penicillin G 0.60

It is of considerable interest to note that the activity of potassium-6-(a-phenoxypropionamido)pencillanate (mixture) is greater than that ofeven the pure abeta isomer in the -S. aureus infection. This activityhas been repeatedly observed. The Table 6 is given CD results from twoother independently run experiments comparing intramuscularlyadministered 6-(a-phenoxypropionamido)penicillanate (mixture) with thepure alpha and beta isomers against S. aureus infection.

TABLE 3.EFFECT OF BACILLUS CEREUS PENICILLINASE ON DIFFE RENTPENICILLINS Percent Inactivation Penicillin I 0 Min. Min. 30 Min. 60Min. 120 Min.

Potassium (Ha-phenoixtpropiona1nido)penicillanate (mix ure) 0 40 56. 88100 Penicillin V. 0 100 100 100 100 Penicillin G 0 81 100 100 100 Acidstability studies TABLE 4.-PERCENT DECREASE IN ACTIVITY MixturePenicillin V Penicillin G T ergp. Hrs.

pH2 pH3 pH2 pH3 p112 pH3 TABLE 6.-CD50 VALUES USING STAPHYLOCOOOUSAUREUS (SMITH) C 50 s/ Penicillin Experiment 1 Experiment 2 Alpha isomer0.72 1.25 Beta isomer 0. 60 0. 70 Mixture 0. 40 0. 45

The compounds of the present invention may be viewed, broadly speaking,as the result of combining the single, naturally occurring opticalisomer, 6-aminopenicillanic acid, with an acid which contains at leastone asymmetric carbon atom as indicated by the asterisk, thus:

Thus, the product (amide or pencillin) when a racemic acid is used willbe a mixture of two diastereoisomers. Both are biologically active andboth of these isomers and mixtures thereof are included within the scopeof the present invention. As illustrated above, the individual isomersare prepared in pure form by starting with the pure dextro or levo formof the acid or by physical separation of the mixture produced from theracernic acid.

23 I claim:

1. The process of preparing an antibacterial composition comprising amixture of the levo and dextro isomers of potassiuma-phenoxyethylpenicillin, said dextro isomer when in pure formdecomposing at about. 230 to 231 C. and exhibiting [011 of about +251(c.=1 percent in water), said levo isomer when in pure form decomposingat about 238 to 239 C. and exhibiting [04 of about +218 (c. l percent inwater), and said isomers being present in said mixture in a weightrelation to one another expressable as 60 to 80% i5% by weight of thelevo isomer and conversely 20 to 40% by weight of the dextro isomerwhich comprises the consecutive steps of adding with agitation tomycelium-free Penicillium chrys ogenum fermentation broth preparedwithout added precursor about five moles of racemic a-phenoxypropionylchloride for each mole of 6-aminopencillanic acid therein, said additionbeing made at a rate slow enough to enable the pH to be maintained atabout 7.5, acidifying said reaction mixture to about pH 2, extractingthe levo and dextro isomers of a-phenoxyethylpenicillin therein intoabout one-half volume of methyl isobutyl ketone,

converting said acid penicillin to the solid potassium salt' by stirringsaid methyl isobutyl ketone solution with concentrated aqueous potassiumacetate butter and collecting said solid salt.

2- In the process of claim 1, the process of preparing an antibacterialcomposition comprising a mixture of the levo and dextro isomers ofpotassium a-phenoxyethylpem icillin, said dextro isomer when in pureform decom-:

posing at about 230 to 231 C. and exhibiting of about +251 (c.=1 percentin water), said levo isomer when in pure form decomposing at about 238to 239 C. and exhibiting [111 of about +218 (c.=l percent in Water), andsaid isomers being present in said mixture in a weight relation to oneanother expressable as 60 to 80% :5% by weight of the levo isomer andconversely to 15% by weight of the dextro isomer which comprises theconsecutive steps of adding with agitation to mycelium-free Penicilliumchrysogenum fermentation broth prepared without added precursor abovefive moles of racemic a-phenoxypropionyl chloride for each mole of6-aminopenicillanic acid therein, said addition being made at a rateslow enough to enable the pH to be maintained at about 7.5 by theaddition of sodium hydroxide, acidifying said reaction mixture toabout'pH 2, extracting the levo and dextro isomers ofa-phenoxyethylpenicillin therein into about one-half volume of methylisobutyl ketone, converting said acid penicillin to the solid potassiumsalt by stirring said methyl isobutyl ketone solution with concentratedaqueous potassium acetate buffer, collecting said solid salt and washingsaid salt with at least one member selected from the group consisting ofmethyl isobutyl ketone, butanol and acetone.

3. In the process of claim 1, the process of preparing an antibacterialcomposition comprising a mixture of the levo and dextro isomers ofpotassium a-phenoxyethylpenicillin, said dextro isomer when in pure formdecomposing at about 230 to 231 C. and exhibiting [041 of about +251(c.=1 percent in water), said levo isomer when in pure form decomposingat about 238 to 239 C. and exhibiting [411 of about+218 (c.=1 percent inWater), and said isomers being present in said mixture in a weightrelation to one another expressable as to i5% by weight of the levoisomer and conversely 20 to 40% :5% by weight of the dextro isomer whichcomprises the consecutive steps of adding with agitation at about 1020C. to mycelium-free Penicillium chrysogeum fermentation broth preparedwithout added precursor about five moles of racemic a-phenoxypropionylchloride for each mole. of -aminopenicillanic acid therein, saidaddition being made at a rate slow enough to enable the pH to bemaintained at about 7.5 by the addition of sodium hydroxide, agitatingsaid reaction mixture at the end of said addition of a-phenoxypropionylchloride for at least'thirty minutes, acidifying said reaction mixtureto about pH 2, extracting the levo and dextro isomers ofa-phenoxyethylpenicillin therein into about one-half volume of methylisobutyl ketone, converting said acid penicillin to the solid potassiumsalt by stirring said methyl isobutyl ketone solution in the cold withconcentrated aqueous potassium acetate buffer, collecting said solidsalt and Washing said salt with at least one member selected from thegroup consisting of methyl isobutyl ketone, butanol and acetone.

No references cited.

NICHOLAS S. RIZZO, Primary Examiner.

1.THE PROCESS OF PREPARING AN ANTIBACTERIAL COMPOSITION COMPRISING AMIXTURE OF THE LEVO AND DEXTRO ISOMERS OF POTASSIUMA-PHENOXYETHYLPENICILLIN, SAID DEXTRO ISOMER WHEN IN PURE FORMDECOMPOSING AT ABOUT 230 TO 231*C. AND EXHIBITING (A)D24 OF ABOUT +251*(C.=1 PERCENT IN WATER), SAID LEVO ISOMER WHEN IN PURE FORM DECOMPOSINGAT ABOUT 238 TO 239*C. AND EXHIBITING (A)C24 OF ABOUT +218* (C.=1PERCENT IN WATER), AND SAID ISOMERS BEING PRESENT IN SAID MIXTURE IN AWEIGHT RELATION TO ONE ANOTHER EXPRESSABLE AS 60 TO 80% $ 5% BY WEIGHTOF THE LEVO ISOMER AND CONVERSELY 20 TO 40% $5% BY WEIGHT OF THE DEXTROISOMER WHICH COMPRISES THE CONSECUTIVE STEPS OF ADDING WITH AGITATION TOMYCELIUM-FREE PENICILLIUM CHRYSOGENUM FERMENTATION BROTH PREPAREDWITHOUT ADDED PRECURSOR ABOUT FIVE MOLES OF RACEMIC A-PHENOXYPROPIONYLCHLORIDE FOR EACH MOLE OF 6-AMINOPENCILLANIC ACID THEREIN, SAID ADDITIONBEING MADE AT A RATE SLOW ENOUGH TO ENABLE THE PH TO BE MAINTAINED AT ABOUT 7.5, ACIDIFYING SAID REACTION MIXTURE TO ABOUT PH 2, EXTRACTING THELEVO AND DEXTRO ISOMERS OF A-PHENOXYETHYLPENICILLIN THEREIN INTO ABOUTONE-HALF VOLUME OF METHYL ISOBUTYL KETONE, CONVERTING SAID ACIDPENICILLIN TO THE SOLID POTASSIUM SALT BY STIRRING SAID METHYL ISOBUTYLKETONE SOLUTION WITH CONCENTRATED AQUEOUS POTASSIUM ACETATE BUFFER ANDCOLLECTING SAID SOLID SALT.